Curtaz Carolin J, Wucherpfennig Sophia, Al-Masnaea Emad, Herbert Saskia-Laureen, Wöckel Achim, Meybohm Patrick, Burek Malgorzata
University Hospital Würzburg, Department of Gynecology and Obstetrics, Würzburg, Germany.
University Hospital Würzburg, Department of Anaesthesiology, Intensive Care, Emergency and Pain Medicine, Würzburg, Germany.
Front Drug Deliv. 2024 Jun 27;4:1331126. doi: 10.3389/fddev.2024.1331126. eCollection 2024.
Therapy of cerebral diseases such as brain metastatic breast cancer is still challenging. Due to the blood-brain barrier (BBB), a tight barrier that protects the brain and prevents the passage of many drugs, therapeutically sufficient drug concentrations in the brain are often not achieved. Therefore, methods and drugs to manipulate the BBB permeability are required. Here we used high-throughput screening (HTS) to identify chemicals that may increase BBB permeability. Human BBB model derived from hematopoietic CD34 stem cells (differentiated to brain-like endothelial cells, BLECs) was used. BLECs were seeded on 96-well plates coated with biotinylated gelatin, treated with respective chemicals for 24 h followed by addition of FITC-avidin for permeability estimation. Selected substances were further tested on BLECs. Cell viability, gene and protein expression were measured using CellTiter-Glo, qPCR and Western blot, respectively. From 1,278 compounds, we identified 175 substances that cause at least a 50 percent increase in BBB permeability. Two substances from the substance classes used in breast cancer therapy, GW2974 (tyrosine kinase inhibitor) and 4-amino-1,8-naphthalimide (ANI) (PARP inhibitor), were analyzed in more detail. ANI was nontoxic to BLECs, while GW2974 decreased or increased viability depending on the concentration used. Both compounds significantly increased BBB permeability and altered protein and mRNA expression in BLECs. Influencing the BBB permeability in patients with brain metastases could increase the response rate to systemic therapy. Using HTS, we were able to accurately and quickly identify compounds that increase BBB permeability and show that using this type of screening method can be applied to endothelial paracellular permeability testing.
脑转移性乳腺癌等脑部疾病的治疗仍然具有挑战性。由于血脑屏障（BBB），这一紧密的屏障保护大脑并阻止许多药物通过，因此大脑中往往无法达到治疗所需的药物浓度。因此，需要操纵血脑屏障通透性的方法和药物。在这里，我们使用高通量筛选（HTS）来识别可能增加血脑屏障通透性的化学物质。使用了源自造血CD34干细胞（分化为脑样内皮细胞，BLECs）的人血脑屏障模型。将BLECs接种在涂有生物素化明胶的96孔板上，用各自的化学物质处理24小时，然后加入FITC-抗生物素蛋白以评估通透性。对选定的物质在BLECs上进行进一步测试。分别使用CellTiter-Glo、qPCR和蛋白质印迹法测量细胞活力、基因和蛋白质表达。从1278种化合物中，我们鉴定出175种物质可使血脑屏障通透性至少增加50%。对乳腺癌治疗中使用的两类物质中的两种，GW2974（酪氨酸激酶抑制剂）和4-氨基-1,8-萘二甲酰亚胺（ANI）（PARP抑制剂）进行了更详细的分析。ANI对BLECs无毒，而GW2974根据所用浓度降低或增加细胞活力。这两种化合物均显著增加血脑屏障通透性，并改变BLECs中的蛋白质和mRNA表达。影响脑转移患者的血脑屏障通透性可能会提高全身治疗的反应率。通过高通量筛选，我们能够准确快速地鉴定出增加血脑屏障通透性的化合物，并表明使用这种筛选方法可应用于内皮细胞旁通透性测试。
